【作者】 邓君萍；

【导师】 陈放；

【作者基本信息】 四川大学 ， 植物学， 2005， 硕士

【摘要】 选择合适的筛选剂及抗生素种类和浓度是利用农杆菌介导转化法进行植物基因工程研究获得转化植株以及较高转化植株的一个重要前提。以麻疯树（Jatropha curcas L.）的子叶、真叶、根及其对应的愈伤组织作为实验材料,研究各种抗生素对其生长的影响。在MS培养基中添加不同浓度和不同种类的抗生素,比较其对麻疯树（Jatropha curcas L.）不同外植体及对应愈伤组织生长的影响。方差分析和总体观察的结果表明,用于筛选转化植株的最佳抗生素是卡那霉素,其最佳抗性筛选浓度应该为10-25mg/L。氨苄青霉素和特美汀是两种抑菌性抗生素,一般的使用浓度为500mg/L,不同菌株有定的差异,使用原则是达到抑菌目的的同时使抗生素对植物愈伤组织形成和分化的影响最少,即尽量降低其浓度。综合分析,本实验中,氨苄青霉素对麻疯树外植体的生长影响较小,实际还需要考虑抑菌效果。 为了构建麻疯树的遗传转化体系,首先应建立其高频再生和高效转化系统,而瞬时表达测试是优化转化系统条件的快速有效的方法。影响瞬时表达的因素很多,本实验主要对四个因素进行测试:预培养天数（1天、2天）、不同激素配比的分化培养（IBA 1.0mg/L+BA 0.4mg/L、IBA 0.2mg/L+BA 2mg/L）、是否加入乙酰丁香酮（+AS、-AS）、外植体类型（子叶、真叶、下胚轴、根）。瞬时表达率=有gus基因瞬时表达的外植体个数/总外植体数,综合外植体瞬时表达的程度,最后得出初步结论:麻疯树（培养35天左右）子叶在分化培养基（IBA 1mg/L,BA0.4 mg/L）上预培养2天,并且在预培养基和离心后的MS₀农杆更多还原

【Abstract】 Important presupposition for selecting proper medium and antibiotics concentration is the plant genetic engineering using Argobacterium tumefaciens.The cotyledon, leaf, root and the corresponding callus of Jatropha curcas L. were selected to be experimental materials to carry out research on effects of different antibiotics concentration on their growth. Different concentrations of various antibiotics were added into MS culture media with hormone combinations and then their influences on the growth of the explants and callus were compared. After variance analysis and overall observation of tissue culture, it was concluded that the best antibiotic for selecting transformed explants and callus was kanamycin and the best resistance selection concentration was between 10~25mg/L. Ampicillin and Tementin are two ablastin. Generally, the concentration of them were 500mg/L. Because the concentration varied in different bacteria, considering the both effect of inhibiting the bacteria and negative influence of inducing callus by antibiotics. We decreased the concentration of antibiotics. As the results, Ampicillin had little influence on explant, and the effect of inhibiting the bacteria should be considered in advance.High-frequency regenerative and transformation system is build tofound genetic transformation system of Jatropha curcas L. transient expression test is the most fastest and effectivest method to optimize the condition of transformation system.Many factors can influence transient expression.The research mainly tests four factors, that is, the number of days of preparative culture (one day, two days), differentiation culture using hormones with different concentration ratios (IBA 1.0mg/L + BA 0.4 mg/L、 IBA 0.2mg/L + BA 2mg/L), with AS or without AS and the type of explants (cotyledon, leaf, dowm hypocotyl and root).The ratio of transient expression is the number of explants that express- instaneously gus gene divides that of all the explants. Concerned with the extent of transient expression of explatns, the results show that the effectiveness of transient expression is rather high(60%) after the cotyledons of Jatropha curcas L. are preparatively cultured in the differentiation culture mediums(IBA 1 mg/L, BA0.4 mg/L) for two days, and AS is added in preparative culture medium and MSo bacterial liquid that has been centrifugated. The extents of colouration are not identical. This may be due to the conformity degrees of T-DNA. Some questions will be resolved, for example, slices are not so ideal that they influence observation and photograph of inner colouration condition, another thing, discoloring time is so long and is not ideal.更多还原